A decade with nucleic acid-based microbiological methods in safety control of foods.

In the last decade, nucleic acid-based methods gradually started to replace or complement the culture-based methods and immunochemical assays in routine laboratories involved in food control. In particular, real-time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry-over contamination. Basic advantages provided by nucleic acid-based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid-based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.

First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry.

The development of a multi-method for the detection of seven allergens based on liquid chromatography and triple-quadrupole tandem mass spectrometry in multiple reaction mode is described. It is based on extraction of the allergenic proteins from a food matrix, followed by enzymatic digestion with trypsin. The chosen marker peptides were implemented into one method that is capable of the simultaneous detection of milk, egg, soy, hazelnut, peanut, walnut and almond. This method has been used to detect all seven allergenic commodities from incurred reference bread material, which was baked according to a standard recipe from the baking industry. Detected concentrations ranged from 10 to 1000 µg/g, demonstrating that the mass spectrometric based method is a useful tool for allergen screening.

Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction.

The design of real-time polymerase chain reaction (PCR) assays for the detection of meat in processed products has focused on using small amplicons, often to the detriment of specificity. However, the relationship between amplification rates and the amplicon size for processed meat products has yet to be determined. To investigate this relationship, real-time PCR assays were designed to give a series of amplicons of increasing size. These assays were then used to assess amplification rates, in relation to amplicon size, in processed meat matrices. Although the most sensitive assays were those that used the smallest amplicons, amplification was still observed using amplicons of 351 base pairs for highly processed samples. It was found, therefore, that although in general, amplicons should be as small as possible, larger amplicons give efficient amplification and that small amplicons should not be chosen if they compromise assay specificity.

The Pisum sativum MAP kinase homologue (PsMAPK) rescues the Saccharomyces cerevisiae hog1 deletion mutant under conditions of high osmotic stress.

Previous analysis of the MAP kinase homologue from Pisum sativum (PsMAPK) revealed a potential MAP kinase motif homologous to that found in eukaryotic cdc2 kinases. Sequence comparison showed a 47% identity on amino acid sequence basis to the Saccharomyces cerevisiae Hog1p MAP kinase involved in the osmoregulatory pathway. Under conditions of salt-stress aberrant morphology of a hog1 deletion mutant was completely restored and growth was partially restored by expression of the PsMAPK. This shows that PsMAPK is functionally active as a MAP kinase in S. cerevisiae. Comparison of PsMAPK with other kinases involved in osmosensitivity, showed a high degree of homology and implicates a possible role for PsMAPK in a P. sativum osmosensing signal transduction pathway.

An RNA transcription-based amplification technique (NASBA) for the detection of viable Salmonella enterica.

Possession of mRNA is indicative of cell viability. RTPCR is not appropriate for mRNA detection as it cannot unambiguously detect mRNA in a DNA background. The alternative amplification technique, NASBA, avoids the disadvantages of RTPCR. We have devised a method for detection of viable Salmonella enterica. This involves NASBA amplification of mRNA transcribed from the dnaK gene. Amplification of mRNA extracted from viable and heat-killed cells from the same population produced consistent and highly significant (P > 0.01) differences between the respective signals. The signal obtained from viable cells was completely eradicated by RNase treatment, while PCR amplification of treated and untreated samples was unaffected, indicating that NASBA was unaffected by background DNA.

Truncation of oligonucleotide primers confers specificity on real-time polymerase chain reaction assays for food authentication.

The advent of real-time polymerase chain reaction has revolutionized the field of molecular biology, but the design and optimization of these assays has been largely overlooked in the literature. This dearth of information is probably in response to the provision of assay design software and robust guidelines issued by the leading manufacturer. However, many applications require highly specific assays with no cross-amplification of non-target DNA and it has been found that the software and guidelines, whilst producing assays of great sensitivity, do not necessarily produce specific assays. Two complementary strategies were used to confer specificity on a real-time assay, first by placing the 3' end of the primers on a point of sequence heterogeneity and, second, by truncating the primers at the 5' end to lower the calculated melting temperature. Using these strategies in concert, a specific assay for a conserved region of the mitochondrial cytochrome b gene was developed that can be used for the unambiguous detection of the target species in a meat mixture. This approach can be used for any real-time polymerase chain reaction assay to increase assay selectivity and specificity.